Application of lactobacillus rhamnosus HPT112 in preparation of medicine for preventing and / or treating hyperuricemia

Abstract

The invention discloses application of lactobacillus rhamnosus HPT112 in preparation of a medicine for preventing and / or treating hyperuricemia, and belongs to the field of microorganisms, the lactobacillus rhamnosus HPT112 is preserved in the China Center for Type Culture Collection on November 20, 2023, and the preservation number is CCTCC NO: M20232273. Tests prove that the lactobacillus rhamnosus HPT112 has the function of regulating bile acid metabolism, so that the uric acid reducing effect is achieved, and the uric acid reducing effect equivalent to or even better than that of an existing hyperuricemia medicine can be achieved. Compared with existing hyperuricemia drugs, the lactobacillus rhamnosus HPT112 has the advantages that toxic and side effects are low, safety is high, a brand-new drug for preventing and / or treating hyperuricemia can be developed by the aid of the lactobacillus rhamnosus HPT112, and accordingly inflammatory reaction caused by hyperuricemia can be prevented and / or treated.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and in particular relates to application of Lactobacillus rhamnosus HPT112 in preparing a drug for preventing and / or treating hyperuricemia. Background Art

[0002] Uric acid is the final product of purine compound metabolism. Uric acid often accumulates in the human body due to the lack of uricase in the human body. Diet and genetic factors can cause the liver to produce too much uric acid, while the kidneys and intestines do not excrete enough uric acid, thereby changing the dynamic balance of uric acid in the human body, leading to increased serum uric acid levels, and thus leading to hyperuricemia. When the human body is simply in a state of hyperuricemia, there are no conscious symptoms, but if this state persists for a long time, urate in the blood will crystallize and deposit in the subcutaneous tissue, kidneys, joints and other parts, and then a series of clinical manifestations such as gout and gout complications will occur. Hyperuricemia has become the second most common metabolic disease after type 2 diabetes, seriously endangering people's lives and health. Many epidemiological studies have shown that the risk of hyperuricemia increases with the increase of serum uric acid levels.

[0003] At present, the following drugs are mainly used for the treatment of hyperuricemia, including: lecithin, benzbromarone, etc. These drugs are used to control the level of uric acid in the blood, thereby preventing and treating hyperuricemia. Among them, lecithin and benzbromarone mainly control uric acid by promoting uric acid excretion. However, these drugs have some adverse reactions to varying degrees. For example, lecithin can cause acute kidney injury, cardiotoxicity, and hepatotoxicity; benzbromarone can cause damage to the gastrointestinal system, hepatobiliary system, urinary system, etc. Moreover, these drugs have greater toxic side effects and lower tolerance at conventional doses.

[0004] Probiotics refer to a class of microorganisms that are beneficial to the human body and are mainly found in the intestines of humans and animals. These microorganisms produce a variety of beneficial effects on the host by regulating the metabolism of intestinal flora. Existing studies have shown that probiotics have the function of regulating metabolic dysfunction in the body. Therefore, regulating bile acid metabolism through probiotics and thus achieving the effect of lowering uric acid may be an effective method for preventing and treating hyperuricemia. This research will provide new ideas and methods for the prevention and treatment of hyperuricemia, which is expected to improve human health and is of great significance to the sustainable development of society. Summary of the invention

[0005] The object of the present invention is to provide the use of Lactobacillus rhamnosus HPT112 in preparing a drug for preventing and / or treating hyperuricemia.

[0006] The technical solution adopted by the present invention to solve the technical problem is as follows:

[0007] The present invention provides application of Lactobacillus rhamnosus HPT112 in preparing a drug for preventing and / or treating hyperuricemia.

[0008] Among them, the Lacticaseibacillus rhamnosus HPT112 was deposited in the China Center for Type Culture Collection on November 20, 2023, with the deposit number being CCTCC NO: M20232273.

[0009] Wherein, the function of Lactobacillus rhamnosus HPT112 is:

[0010] (1) Reduce the levels of uric acid, urea nitrogen and creatinine in serum;

[0011] (2) reduce the total bile acid content in feces;

[0012] (3) reduce the levels of taurocholate, glycocholate, chenodeoxycholic acid, deoxycholate, taurodeoxycholate, and tauroursodeoxycholate in cecal contents;

[0013] (4) Regulate the gene expression level of urate transporter in the mouse intestine.

[0014] Furthermore, the urate transporters are: OAT10, GLUT9a, GLUT9b and OCTN2.

[0015] Furthermore, the Lactobacillus rhamnosus HPT112 exists in the form of a bacterial suspension.

[0016] Furthermore, the preparation method of the bacterial suspension is:

[0017] Lacticaseibacillus rhamnosus HPT112 was inoculated into liquid MRS medium, then statically cultured and centrifuged to discard the supernatant to obtain bacterial precipitate; the bacterial precipitate was prepared into a bacterial suspension with sterile physiological saline, and the bacterial count was adjusted to 1.0×10 9 CFU / mL.

[0018] The beneficial effects of the present invention are:

[0019] 1. The present invention provides the use of Lactobacillus rhamnosus HPT112 in the preparation of a drug for preventing and / or treating hyperuricemia. Experiments have shown that Lactobacillus rhamnosus HPT112 has the function of regulating bile acid metabolism, thereby playing a role in lowering uric acid, and can achieve a uric acid-lowering effect comparable to or even better than that of existing hyperuricemia drugs.

[0020] 2. The Lactobacillus rhamnosus HPT112 used in the present invention is a probiotic. Compared with existing hyperuricemia drugs, Lactobacillus rhamnosus HPT112 has lower toxicity and side effects and higher safety, and can be used to prevent and / or treat hyperuricemia.

[0021] 3. The present invention has found through scientific experiments that Lactobacillus rhamnosus HPT112 can reduce the levels of uric acid, urea nitrogen, and creatinine in serum, reduce the total bile acid content in feces, reduce the levels of taurocholate, glycocholate, chenodeoxycholic acid, deoxycholate, taurodeoxycholate, and tauroursodeoxycholate in the cecal contents, and regulate the gene expression level of urate transporter in the mouse intestine; it can effectively reduce the uric acid level in serum, thereby preventing and treating hyperuricemia; using Lactobacillus rhamnosus HPT112, a new drug for preventing and / or treating hyperuricemia can be developed, thereby preventing and / or treating the inflammatory response caused by hyperuricemia. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 The uric acid, urea nitrogen and creatinine levels in the serum of each group of rats in the present invention are shown in the figure. In the figure, A, uric acid content; B, urea nitrogen content; C, creatinine content.

[0023] Figure 2 is the total bile acid content in the feces of each group of rats in the examples of the present invention.

[0024] Figure 3 Figure 2 shows the changes in bile acid metabolites of rats in each group in the examples of the present invention.

[0025] Figure 4 The relative contents of primary bile acid and secondary bile acid in each group of rats in the examples of the present invention are shown in the figure. In the figure, A, relative abundance of primary bile acid biosynthesis; B, relative abundance of secondary bile acid biosynthesis.

[0026] Figure 5 The mRNA expression levels of urate pathway proteins in each group of rats in the present invention are shown in the figure. In the figure, A, OAT10; B, GLUT9a; C, GLUT9b; D, OCTN2. DETAILED DESCRIPTION

[0027] The present invention discloses the use of Lactobacillus rhamnosus HPT112 in the preparation of a drug for preventing and / or treating hyperuricemia. Those skilled in the art can refer to the content of this article and appropriately improve the process parameters to achieve it. It is particularly important to point out that all similar replacements and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention. The method and application of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0028] The present invention will be further described in detail below in conjunction with specific examples. The following examples are not intended to limit the present invention but are merely intended to illustrate the present invention.

[0029] Unless otherwise specified, the experimental methods used in the following examples are generally carried out under conventional conditions. The materials, reagents, etc. used in the following examples are all commercially available unless otherwise specified.

[0030] In the following examples, the source information of some products is as follows:

[0031] Lacticaseibacillus rhamnosus HPT112 was deposited in the China Center for Type Culture Collection, abbreviated as CCTCC, on November 20, 2023. The address is: Wuhan University, No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province (Wuhan University Collection Center), and the preservation number is CCTCC NO: M20232273.

[0032] In addition, other information about Lacticaseibacillus rhamnosus HPT112 including isolation, identification, preservation, etc. can be referred to Chinese patent publication number CN117701447A (the invention name is Lactobacillus rhamnosus HPT112 and its use in preparing antihypertensive drugs).

[0033] Example: Preparation of Lactobacillus rhamnosus HPT112 bacterial suspension

[0034] Lactobacillus rhamnosus HPT112 was inoculated into liquid MRS medium, cultured for 20 h, centrifuged at 3000 r / min and 4 °C for 10 min, and the supernatant was discarded to obtain bacterial precipitate. The bacterial precipitate was then prepared into a bacterial suspension with sterile saline, and the bacterial count of the bacterial suspension was adjusted to 1.0 × 10 according to its OD600 absorbance and plate count results.9 CFU / mL.

[0035] Test example: Effects of Lactobacillus rhamnosus HPT112 on hyperuricemia model rats

[0036] 1. Establishment and grouping of animal models;

[0037] SPF grade SD male rats (8 weeks old) were purchased from Changchun Yisi Experimental Animal Technology Co., Ltd. The breeding environment was 23±1℃ and free access to water. SPF grade SD male rats were randomly divided into 3 groups after one week of adaptation, with 10 rats in each group. The specific grouping is as follows:

[0038] ① Normal diet group (ND group): normal diet (basic feed, purchased from Changchun Yisi Experimental Animal Technology Co., Ltd.), 200 μL 0.01M PBS was gavaged per day for 20 weeks;

[0039] ② Hyperuric acid model group: high fructose and high fat diet (the formula of high fructose and high fat diet is: 61% normal breeding mouse feed, 20% lard, 19% fructose), 200 μL 0.01M PBS was gavaged per day for 20 weeks;

[0040] ③ Lactobacillus rhamnosus group (HPT112 group): high fructose and high fat diet (the formula of high fructose and high fat diet is: 61% normal breeding feed, 20% lard, 19% fructose), 200 μL 1.0×10 9 CFU / mL of Lactobacillus rhamnosus HPT112 bacterial suspension for a total of 20 weeks.

[0041] 2. Effects of Lactobacillus rhamnosus HPT112 on the levels of uric acid, urea nitrogen and creatinine in the serum of rats with hyperuricemia model;

[0042] After the 20th week of feeding, blood was collected from the eyeballs of the rats. The rats were deprived of food for 12 hours before blood collection. The collected blood was placed in a refrigerator at 4°C overnight; then centrifuged at 3000rpm for 10 minutes, the upper serum was collected, and the levels of uric acid (UA), urea nitrogen, and creatinine in the serum of each group of rats were detected using the kit of Nanjing Jiancheng Bioengineering Institute.

[0043] Conclusion: Due to the high fructose and high fat diet, the uric acid content (μmol / L) in the serum of the rats in the Model group was significantly different from that in the ND group (P<0.01). Figure 1 As shown in Figure A, the hyperuricemia model rats were successfully established. At the same time, the urea nitrogen content (mg / L) in the serum of the rats in the Model group was significantly different from that in the ND group (P<0.01). Figure 1As shown in B; the creatinine content (μmol / L) in the serum of rats in the Model group was significantly different from that in the ND group (P<0.01). Figure 1 As shown in C. After 20 weeks of intervention with Lactobacillus rhamnosus HPT112, the levels of uric acid, urea nitrogen, and creatinine in the serum of hyperuricemia model rats were significantly reduced (P<0.01). Figure 1 As shown in A, B, and C.

[0044] 3. Effect of Lactobacillus rhamnosus HPT112 on total bile acid in feces of rats with hyperuricemia model;

[0045] Bile acid is an important component of bile and plays an important role in metabolism. Bile acid is mainly present in the enterohepatic circulation system and plays a certain protective role through recycling. Bile acid plays a vital role in the digestive system. Excessive bile acid entering the intestine will cause the intestinal barrier to decrease, leading to inflammatory response and causing metabolic dysfunction.

[0046] In this example, feces of rats in each group were taken and the total bile acid (TBA) content of rats in each group was detected using a kit from Nanjing Jiancheng Bioengineering Institute.

[0047] Conclusion: After 20 weeks of intervention with Lactobacillus rhamnosus HPT112, the total bile acid content in the feces of rats in the HPT112 group was significantly reduced, about 31.65% lower than that in the Model group. Figure 2 shown.

[0048] 4. Effects of Lactobacillus rhamnosus HPT112 on bile acid metabolites in the cecum of hyperuricemia model rats;

[0049] 15 mg of rat cecal contents were homogenized with a small amount of ultrapure water; ultrasonic extraction was then performed using a mixture of methanol and acetonitrile in a volume ratio of 1:1; and then the results were detected on a Thermo Vanquish (Thermo Fisher Scientific, USA) ultra-high performance liquid chromatography system, while ACQUITY HSS T3 column (2.1×100 mm, 1.8 μm) (Waters, Milford, MA, USA) with a flow rate of 0.3 mL / min; Thermo OrbitrapExploris 120 mass spectrometer (Thermo Fisher Scientific, USA), electrospray ionization (ESI) source and positive and negative ion modes were used for mass spectrometry data acquisition. LC-MS / MS metabolomics analysis was used to detect the changes of various bile acid metabolites in the cecal contents of rats in each group.

[0050] Conclusion: High fructose and high fat diet led to a significant increase in the contents of chenodeoxycholate and glycochenodeoxycholate in primary bile acids and deoxycholate, taurodeoxycholate and glycodeoxycholate in secondary bile acids, while the contents of taurocholate, glycocholate, chenodeoxycholate in primary bile acids and deoxycholate, taurodeoxycholate and tauroursodeoxycholate in secondary bile acids decreased after intervention with Lactobacillus rhamnosus HPT112. Figure 3 shown.

[0051] like Figure 4 As shown, the biosynthesis of primary and secondary bile acids in the intestinal microbiota of rats in each group was significantly upregulated.

[0052] Therefore, these findings indicate that a high-fructose, high-fat diet disrupts the balance of intestinal bile acids in hyperuric acid model rats, and Lactobacillus rhamnosus HPT112 can restore the disordered bile acid metabolism.

[0053] 5. Effects of Lactobacillus rhamnosus HPT112 on the uric acid metabolic pathway in hyperuricemia model rats;

[0054] Total RNA was isolated from the small intestinal tissue of each group of rats using TRIpure reagent (Beijing Aidelai Biotechnology Co., Ltd.) according to its instructions, and quantified using an ultraviolet spectrophotometer; reverse transcription and qRT-PCR were performed using glyceraldehyde-3-phosphate dehydrogenase (Gapdh) as an internal reference, and the relative expression of genes was calculated using the 2-ΔΔCt method. The primer sequence information used is shown in Table 1.

[0055] Table 1 Primer sequences

[0056]

[0057] By detecting the gene regulation levels of urate transport-related proteins (reabsorption and secretion) in the intestines of each group of rats, the results are as follows Figure 5As described above, it was found that a high-fructose, high-fat diet caused an increase in the mRNA levels of organic anion transporter (OAT10) and glucose transporter 9 (GLUT9a and GLUT9b) in the intestines of rats, while the mRNA level of organic cation transporter 2 (OCTN2) decreased. After 20 weeks of intervention with Lactobacillus rhamnosus HPT112, the levels of each gene were significantly regulated. This shows that Lactobacillus rhamnosus HPT112 can effectively regulate the gene expression level of urate transporter in the intestines of each group of rats.

[0058] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein, but these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. Use of Lactobacillus rhamnosus HPT112 in the preparation of a drug for preventing and / or treating hyperuricemia.

2. The use according to claim 1, characterized in that: The Lacticaseibacillus rhamnosus HPT112 was deposited in the China Center for Type Culture Collection on November 20, 2023, with the deposit number being CCTCC NO: M20232273.

3. The use according to claim 1, characterized in that: The function of the Lactobacillus rhamnosus HPT112 is: (1) Reduce the levels of uric acid, urea nitrogen and creatinine in serum; (2) reduce the total bile acid content in feces; (3) reduce the levels of taurocholate, glycocholate, chenodeoxycholic acid, deoxycholate, taurodeoxycholate, and tauroursodeoxycholate in cecal contents; (4) Regulate the gene expression level of urate transporter in the mouse intestine.

4. The use according to claim 3, characterized in that: The urate transporters are: OAT10, GLUT9a, GLUT9b and OCTN2.

5. The use according to claim 1, characterized in that: The Lactobacillus rhamnosus HPT112 exists in the form of bacterial suspension.

6. The use according to claim 5, characterized in that: The preparation method of the bacterial suspension comprises: inoculating Lactobacillus rhamnosus HPT112 into a liquid MRS culture medium, then performing static culture, centrifuging and discarding the supernatant to obtain a bacterial precipitate; preparing the bacterial precipitate into a bacterial suspension with sterile physiological saline, and adjusting the bacterial count to 1.0×10 9 CFU / mL.

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